Contrasting Photosensitized Processes of Ru(II) Polypyridyl Structural Isomers Containing Linear and Hooked Intercalating Ligands Bound to Guanine-Rich DNA

The DNA binding and cellular uptake of the lambda enantiomer of two bis-tetraazaphenanthrene (TAP) Ru(II) polypyridyl complexes containing either a linear dppn (1) or a hooked bdppz (2) benzodipyridophenazine ligand are reported, and the role of different charge-transfer states of the structural isomers in the photo-oxidation of guanine is explored. Both complexes possess characteristic metal-to-ligand charge-transfer (MLCT) bands between 400 and 500 nm and emission at ca. 630 nm in an aerated aqueous solution. Transient visible absorption (TrA) spectroscopy reveals that 400 nm excitation of 1 yields a dppn-based metal-to-ligand charge-transfer (MLCT) state, which in turn populates a dppn intraligand (3IL) state. In contrast, photoexcitation of 2 results in an MLCT state on the TAP ligand and not the intercalating bdppz ligand. Both 1 and 2 bind strongly to double-stranded guanine-rich DNA with a loss of emission. Combined TrA and time-resolved infrared (TRIR) spectroscopy confirms formation of the guanine radical cation when 2 is bound to the d(G5C5)2 duplex, which is not the case when 1 is bound to the same duplex and indicates a different mechanism of action in DNA. Utilizing the long-lived triplet excited lifetime, we show good uptake and localization of 2 in live cells as well as isolated chromosomes. The observed shortening of the excited-state lifetime of 2 when internalized in cell chromosomes is consistent with DNA binding and luminescent quenching due to guanine photo-oxidation.

of 10 kHz, which is split to generate the pump and probe pulses.The pump pulses (1 µJ, 50 fs pulse duration) for ps-TRIR and ps-TrA experiments were generated by second (400 nm) harmonic generation in a β-barium borate (BBO) crystal.
To generate the mid-infrared probe light for TRIR measurements, the 800 nm output was used to pump a white-light continuum-seeded optical parametric amplifier (OPA) (Light Conversion, TOPAS).
The generated signal/idler outputs were then difference frequency mixed by an AgGaS2 crystal producing output pulses of ca.400 cm -1 bandwidth in the mid-infrared region of the spectrum with ca.50 fs pulse duration.To further increase the signal-to-noise ratio for the TRIR measurement, the IR probe beam was further split by a germanium beamsplitter, with one beam forming the reference probe, detected by a 64-pixel mercury cadmium telluride (MCT, IR Associates) detector, this reference probe allows constant monitoring and correction for fluctuations in the probe spectrum and intensity during data acquisition.The second beam is focused on to the sample and recorded on two 128pixel MCT detectors.The output of the two 128-pixel detectors was then spliced during data processing to create a single compete spectrum.
The probe pulse for TrA measurements is generated by focusing part the 800 nm output beam through a white-light continuum generator (CaF2 plates).This white light is then dispersed through a grating monochromator after passing through a 400 nm notch filter, to remove scatter from the excitation beam.The TrA spectrum was then recorded using a 512-pixel silicon detector (Quantum Detectors).
The delay between pump and probe pulses in these experiments was achieved using an optical delay line.

ps to ms time domain experiments:
The Time-Resolved Infrared (TRIR) measurements on the time range from ps to ms were performed on LIFEtime apparatus at the STFC Central Laser Facility. 1 The LIFEtime setup is driven by the two Yb:KGW amplifiers (PHAROS, Light Conversion, each amplifier operating at 100 kHz).The two amplifiers are optically synchronised by sharing the same oscillator operating at 80 MHz repetition rate.One amplifier is driving a single 515 nm pumped OPA (ORPHEUS HP, Light Conversion) which is capable to generate the output beam across 210 -2600 nm range which used as a pump source in TRIR experiment.The pulse picker built into each PHAROS amplifier provides a simple way to adjust the repetition rate of pump pulses, which was set to 2 kHz in the present work.The wavelength of the pump beam was set to 400 nm in the present work, with the pulse energy at sample and the pump spot size to 500 nJ and 120 µm, respectively.The second amplifier was used to pump simultaneously two mid-IR OPA (ORPHEUS ONE and LYRA DFG unit, Light Conversion) generating two mid-IR probe beams for the TRIR experiment.The probing was performed at 100 kHz repetition rate.The pump and two probe beams were overlapped at the sample, and the two probe beams were subsequently sent to the home-built grating spectrographs and two individual probe spectra detected with 128-pixel MCT detectors each (IR Associates).The time delay

S-4
between the pump and the probe beams was achieved using the combination of the optical delay line (for the delay range fs to 12 ns), and oscillator round trip timing to achieve steps of 12 ns.The train of probe pulses coming at 100 kHz covers the time range from 10 µs and longer.The relative polarisations of the pump beam with respect to the two probes was set at "magic" angle.The sample was rastered in the X and Y to minimise sample degradation in the pump beam.The LIFEtime setup offers < 200 fs temporal resolution along with > 400 cm -1 combined spectral window from the two probe OPA's.
All spectra were processed using the in-house Ultraview software provided by the Central Laser Facility (CLF).Spectra were calibrated from pixels into wavenumbers using the Ultracal software.The metal complex and DNA region (1250-1860 cm -1 ) was calibrated by fitting to the absorption bands of polystyrene.All experiments were carried out at 298 K and samples were checked before and after the experiment by UV-Visible spectroscopy (PerkinElmer lambda 950 spectrophotometer) and FTIR in a Nicolet Avatar spectrometer.

Figure S3 :
Figure S3: (a) UV-Vis spectrum of the Δ-1 and Λ-1 enantiomers in water and CD spectrum of the enantiomers (b) summary of the optical properties.(c) UV-Vis spectrum of the Δ-2 and Λ-2 enantiomers in water and CD spectrum of the enantiomers (d) summary of the optical properties.

Figure S13 :
Figure S13: Steady state confocal images of live CHO cells labelled with DAPI and complex 2. Excitation at 405 nm, emission in blue channel (Hoechst stain) and red channel (long pass 650 nm) (a) Confocal overlay of transmission light, DAPI and complex 2 channels.(b)-(c) PLIM images of the same field of view of 2 and CHO cells after 24-hour incubation period.DAPI was allowed to label for only 15 min.

Figure S14
Figure S14 Confocal and PLIM imaging of isolated nuclei and chromosomes incubated with complex 2 at room temperature.(a) and (b) confocal image of isolated nuclei and chromosomes incubated for 24 hr with 50 M of 2 (60 x optical zoom) (c) and (d) PLIM channel (λex = 405 nm/ λdetection = 450 nm long pass).

Figure S16 .Figure S17 :
Figure S16.Region of interest (ROI) lifetime distributions extracted from mid-point of cell recorded in a 2D confocal PLIM slice from 3D imaging (S15, D) of 2 labelled live HeLa cells.405 nm excitation 610 nm long pass filter.